Phosphorylated phosphatidylinositols (phosphoinositides) are a type of membrane bound phospholipid that contributes to multiple diverse processes required during platelet activation. PhosphatidylInositol Transfer Proteins (PITPs) are a small family of proteins that have been demonstrated in vitro to bind and transfer phosphoinositide monomers from one cellular compartment to another in an energy independent manner during vesicle trafficking and phospholipid signaling. Although there are no studies on the role of PITPs in hematopoietic cells, there is evidence in yeast cells that these proteins are essential for the biosynthesis and metabolism of phosphoinositides. Platelets have two dominant PITP family members, PITP1 and PITP2. The overall hypothesis of this proposal is that these individual PITP isoforms have non- overlapping functions that are each essential for the generation and spatial localization of discrete species of phosphoinositides within platelets. A secondary hypothesis is that the enzymatic activities of both PITP isoforms are necessary for normal platelet adhesion, aggregation, and granule secretion. To understand the unique and discrete roles of these individual PITP isoforms in platelet biology, we have generated mice containing conditional null mutations within the PITP1 and PITP2 genes. This R01 is to request funds that will allow us to characterize PITP1fl/fl PF4Cre+, PITP2fl/fl PF4Cre+, and PITP1fl/fl PITP2fl/fl PF4Cre+ (double knockout) mice. These mice have platelets and mature megakaryocytes lacking either PITP1 or PITP2, but they have normal expression of these proteins in all other tissues. Loss of either isoform results in thrombocytopenia. Our preliminary data indicates that platelets lacking PITP1 have a complete loss in the second messenger, Ins(3,4,5)P3 (also known as IP3) following stimulation by maximal doses of thrombin. I plan to perform a comprehensive and systematic study of the function of these individual PITP isoforms in platelets in order to understand their unique biochemical role and biologic importance within platelets. In Aim 1, we will determine the link between PITP isoforms and polyphosphoinositide synthesis, as well as analyze the contribution of PITPs to platelet signaling. In Aim 2, I propose experiments designed to understand the distinct biochemical functions of the individual PITP isoforms. In the final Aim, we will identify the role of PITP isoforms in platelet activation ex vivo and in vivo. PUBLIC HEALTH RELEVANCE: Heart disease and strokes are among the leading causes of morbidity and mortality in this country. Activated blood platelet cells can form clots at sites of atherosclerosis, and are often the precipitating event for both of these diseases. This work is focused on two platelet proteins, PITP1 and PITP2. I believe that a better understanding of the events that regulate platelet activation, including those mediated by PITP1 and PITP2, will lead to new therapeutic approaches to prevent vascular occlusion, as well as lead to a better understanding of platelet biology.